Biosystematic studies of genus Withania Pauquy in Egypt

Withania (Solanaceae, Solanoideae) is a widespread genus. Comparative macro-, micro-morphological, anatomical, and molecular features of this genus in Egypt were examined using light and scanning electron microscopy to reassess the conflicted taxonomic relationships between the two studied species. The most significant morphological differences that have been found were: the shape of the lamina, apex, anther, and stigma, and the ratio of calyx tube/lobe; anatomical examination of taxonomic interest are as follows: number of vascular bundles, presence of ears and distribution of accessory vascular bundles in petiole and shape of spongy cells, and number of lower parenchyma in the midrib region of the leaf; trichomes of both species showed no significant differences; pollen, and seed characters are of taxonomic significance in differentiation and characterization between them. Protein profiling revealed that W. somnifera has only conserved proteins, while W. obtusifolia possessed both conserved and additional proteins in their SDS-PAGE banding patterns. Eleven starts codon-targeted (ScoT) primers were applied and produced 96 amplicons with an average of 70.83% polymorphism/primer. W. obtusifolia generated more polymorphic bands and maintained monomorphic ones. SDS-PAGE disclosed that both Withania species were 50% related. While Scot-Dendrogram revealed that both Withania species were poorly related. So, protein and molecular analyses showed considerable genetic variations between these two species.


Anatomical investigations
Sections of the vegetative organs (stem, petiole, and leaves) were chosen from fresh material.All assessments were made on all plants at similar developmental stages (fruiting stages) and in comparable positions on each plant.Samples were taken from 4th internodes from the apex about 2-3 (cm) and then fixed in FAA (Formalinglacial acetic acid-70% ethyl alcohol, 5:5:90 V/V).After 24-h fixation, the specimens were transformed into ethyl alcohol series and then embedded in paraffin wax.The specimens were sectioned by a rotary microtome at 10-15 μm; sections were dehydrated in alcohol-xylol series.Sections were stained by safranin and light green according to 71 .The anatomical characters were examined with a Zeiss light stereomicroscope and photographed with a digital camera (OPTIKA).A planimeter was used for the estimation of the percentage of each tissue to the total section area.Terminology according to [72][73][74] .

SDS-PAGE of soluble whole-cell proteins
Protein extractions were performed on 100 mg of frozen tissues collected in triplicate from two Withania plants (W.obtusifolia and W. somnifera).Each sample was ground separately in liquid nitrogen before being mixed with 300 µl of saline and properly vortexed for 30 s.Ten µl of each homogenate was mixed with 20 µl of buffer [10% Sodium Dodecyl Sulfate (SDS), 20% Glycerol, 0.2 M Tris (pH6.6), 10 mM beta-mercapto-ethanol, and 0.05% bromophenolblue] and incubated for 5 min in a 95 °C water bath.Both samples were centrifuged at 13,000 × g for 5 min and kept on ice until it is used.
Electrophoresis was accomplished in a vertical slab mold filled with [25 mM Tris-Hcl, 200 mM glycine, and 0.1% (w/v) SDS] running buffer at 80 V for 4 h.After the run, the gel was stained with Coomassie solution for 20 min with agitation to recognize the protein bands.Coomassie solution was composed of 50% distilled water, 40% methanol, 10% glacial acetic acid, and 0.1% Coomassie brilliant blue.The gel was finally destained with a mixture of distilled water, methanol, and glacial acetic acid in a ratio of 5:4:1, respectively, then photographed and stored.

Molecular analyses
Whole genomic DNA was extracted from 50 mg of frozen tissues collected in triplicate from the two Withania plants using the CTAB (Cetyltrimethylammonium bromide) extraction method of 76 .CTAB buffer comprised 1M Tris HCl (pH 8.0), 5 M NaCl, 0.5 M EDTA, and 20 g CTAB.Polyvinylpyrrolidone and β-mercaptoethanol were freshly mixed into the buffer before extraction.Each tissue was ground separately using liquid nitrogen before mixing with 500 µl CTAB extraction buffer.Each homogenate was incubated for 3 h in a 55 °C water bath.Then, 1.5 µl RNaseA was added to both samples and left for 15 min at 37 °C.At room temperature, chloroform (500 µl) was added to both samples, mix gently, and centrifuged at 16000×g for 7 min.Cold ammonium acetate (7.5 M) and cold isopropanol (1:6, v/v) were added to the aqueous phases of both samples.The samples were gently inverted several times, incubated on ice for 30-40 min, and centrifuged at 16000 xg for 3 min.Throw away the supernatant carefully and washed the pellets with 700 µl (70%) ethanol.The tubes were centrifuged at 16000 xg for 1 min, then the supernatants were poured and the pellets were air dried.Finally, the pellets were re-suspended with 50 µl (1×) TE buffer [10 mM Tris-HCl (pH 8.0), 0.1 mM EDTA].
PCR amplifications were performed using eleven start codon-targeted (Scot) primers, Table 2, according to the methods of 76 with minor modification.In an iced PCR tube, the reaction mixture comprised 12.5 µl DreamTaq Green PCR Master Mix (2×), 2µl primer, and 1µl (50 ng) template DNA forming a 25 μl total volume.The amplification was achieved using Veriti 96-Well Thermal Cycler: initial denaturation of 5 min at 95 °C; 40 cycles of 1 min denaturation at 95 °C, 1 min annealing at 56 °C and 2 min extension at 72 °C; and a final elongation step at 72 °C for 10 min.PCR products were separated by electrophoresis (3 hours, 80 v) through 1.5 % (m/v) agarose gel in 1× TAE buffer (40 mM Tris base, 20 mM acetic acid, and 1 mM EDTA, pH 8.0).Band sizes were visualized and evaluated using Gel-Documentation (G: BOX) (SYNGENE model 680XHR, UK) based on a 3000 bp DNA ladder.

Protein and DNA analyses
The BioRad Gel documentation system (Image lab V6.1.0build 7) provided image analysis software for protein and DNA analyses.MvSP software (V3.22) [www.KoVco mp.com] was used to create a phylogenetic tree using the nearest neighbor with Jaccard's coefficient, and PyElph (V1.3) was used to identify the similarity matrix between the plants by rating the presence (1)/absence (0) of each amplicon.W. somnifera: Leaf broadly ovate with acute apex and attenuate base.Inflorescence 7-10 flowers, calyx tube shorter than lobe, calyx lobe deltoid-lanceolate.The Corolla tube is shorter than the lobe.Filament with cushions at the base.Stigma bilobed.Fruit bright red with 25-30 seeds and seed diameter (2.3-) 2.5-3 × 1.9-2.5 mm.The detailed morphological variations are listed in Table 3.
The morphological variations such as the shape of the leaf and the number of seeds were reported between W. obtusifolia and W. somnifera by 41,77 .The current investigation validated their results; leaf ovate-elliptic with obtuse apex and truncate base in W. obtusifolia while leaf broadly ovate with acute apex and attenuate base in W. somnifera, each berry has 23-25 seeds in W. obtusifolia and 25-30 seeds in W. somnifera.In addition to the previously mentioned feature, there are newly employed characters to distinguish between the Withania species under study, such as calyx lobe shape; ovate in W. obtusifolia even though deltoid-lanceolate in W. somnifera, calyx tube/lobe ratio; tube longer than lobe in W. obtusifolia while tube shorter than lobe in W. somnifera, hairiness of the interior side of the petal lobe; smooth in W. obtusifolia while hairy in W. somnifera, filament without cushions at the base in W. obtusifolia, while with cushions in W. somnifera, anther oblong in W. obtusifolia, ovate in W. somnifera and seed; 1.8-2.2× 1.6-2 mm diameter in W. obtusifolia while (2.3-) 2.5-3 × 1.9-2.5 mm in W. somnifera.Ultimately, the morphological characteristics under examination are distinguishable and diagnostic between the Withania species under study.

Micro-morphology
Pollen Pollen features through LM and SEM are a good source of taxonomic information that can help the species and genera delimitation, and identification and strengthen their systematic position 78 , usually tricolporate rarely 4-colporate in Solanaceae 47 .
In W. obtusifolia, the pollen class type is tricolporate, sub-spheroidal in shape, with a bridge in the middle of the colpus, while in W. somnifera, the pollen class type is tricolporate and tetracolporate, prolate in shape, with or without a bridge in it, and the bridge at the middle or lateral position (Table 3 and Fig. 3).
Pollen morphology of the studied species shows considerable variation in exine pattern which agreed with 34 .Significant pollen characteristics are important in differentiating the investigated Withania species; pollen shape subspheroidal in W. obtusifolia even though prolate in W. somnifera, shape in equatorial view; spheroidal in W. obtusifolia while oblong in W. somnifera, colpus length/width ratio; 9-20 in W. obtusifolia while 14.5-31.3 in W. somnifera.Additional characteristics, such as polar and equatorial diameter, shape in polar view, exine surface, bridge prescience and position, reveal overlaps between the species under study.

Seeds
The seed characters of the genus Withania were investigated using SEM.In both species, the seed shape is quite stable, reniform to sub-reniform with apical not protruding concave (sunken) hilum.The gross overall seed coat   www.nature.com/scientificreports/sculpture is reticulate.The shape of epidermal cells is irregular and not isodiametric, the cells near the margin are smaller than the others.The walls of anticlinal cells are broadly and evenly thickened, with a sinuate cell margin.W. obusifolia confined to Sinai, Gebel Elba characterized by rather small seeds (1.6)1.8-2.2 × 1.6-2(× 10 3 ) mm drying to brown with polygonal (7-10-gonal) or oblong large cells.The basal portion of the anticlinal wall (lumen) was relatively thick with characteristic holes, smooth over the entire inner surface of the cell, and a broad ribbon-like appendage, finely papillate at the summit of the cell.While the cosmopolitan W. somnifera is characterized by large seeds (2.3-) 2.5-3 × 1.9-2.5 (× 10 3 ) mm drying to brown with polygonal (5-6-gonal) small cells.The basal portion of the anticlinal walls is strongly thickened, the thickness covering the whole bottom, densely papillate, and fibrils present over the lateral inner surface of the cells.At the summit of the cell slender ribbon-like appendages were observed in Table 3 and Fig. 4.
The variability in seed surface patterns is seemingly very useful in the recognition of the studied species 79,80 .SEM analysis of the seeds in the present study showed that W. somnifera had a deeply protruding hilum, whereas W. obtusifolia had a shallow, slightly protruding hilum, In W. obtusifolia, the relief of the cell boundary is narrow and ribbon-like, with small papilla at the cell's summit; in W. somnifera, it is broad and densely papillate, outer periclinal wall is concave and has characteristic perforations through the thickening and lateral wall without fibrils in W. obtusifolia while the bottom area is covered by concave thickenings and lateral wall with fibrils in W. somnifera.In brief many spermodermal features are proven to be the diagnostic features that can be used to distinguish Withania species which agree with 80 .

Stem anatomy
In cross-sections, the outline of the stems is more or less circular to angular, with moderate-densely hairy.The epidermis is composed of a single layer of isodiametric-radially elongated cells covered by a very thin wavy cuticle.The cortex differentiated into 4-5 outer collenchymatous composed of isodiametric-tangentially elongated cells, followed by inner parenchymatous with isodiametric cells, filled with sand crystals.The vascular bundles are bicollateral with outer and inner phloem.Xylem with 2-5 vessels mainly in radial groups of 5-7 arches.A large central pith, composed of 16-25 layers with hexagonal and mostly isodiametric parenchymatous cells present in the center, occasionally sand crystals are present (Table 3 and Fig. 5).

Petiole anatomy
The outline is more or less oval with a shallow indentation in the adaxial portion.Epidermis formed of minute radially elongated cells, covered with an evident undulating cuticle and glandular, non-glandular (multicellular, and dendritic) hairs.Stomatal pores on both epidermal tissue facing loose parenchyma.The cortex is differentiated into outer and inner regions; the outer collenchymatous layers are formed of isodiametric-radially elongated cells and inner parenchymatous layers with radially-irregular cel.The vascular system is represented by a central elongated, arc-shaped bi-collateral vascular bundle, in which the xylem arches are ± 35, each in 2-6 series, in addition to 1-4 accessory vascular bundles at each end of the main vascular bundle (Table 4 and Fig. 6).
In W. obtusifolia, the midrib has a broad, dome-shaped adaxial part and a tangentially extended semicircular abaxial part.The vascular system consists of a broad boat-shaped bicollateral strand of xylem band and small broken patches of phloem outside xylem arc.The size of the vascular is 372-435 μm thick.But in W. somnifera the midrib has a broad adaxial hump and still broader, semicircular abaxial part and continuous phloem outside the xylem.The size of the vascular is 506-538 μm thick.Leaf anatomy is bifacial, dorsiventral with a u-shaped midrib region; upper epidermal cell isodiametricradially elongated and covered with wavy cuticle and glandular, non-glandular (multicellular, and dendritic) trichomes.Collenchyma1-2 layers, radially elongated cells.Parenchyma tissue 6-9 layers, iso-radially elongated cells.The main vascular bundle is bicollateral; phloem 4-7 layers upper and lower xylem region and the number of xylem arches is 32-36 each with 3-6 vessels.
Under the main vascular bundles, 5-9 layers of parenchymatous tissue with isodiametric radially elongated cells.Followed the parenchymatous tissue, 1-3 collenchyma layers with isodiametric-radially elongated cells.The parenchyma that faces the stomatal pore has wide cellular space.Lower epidermal tissue isodiametric-radially elongated cells covered with wavy cuticle.In the wings region, the mesophyll is distinguished into palisade and spongy tissues, palisade 1 layer.Spongy tissue has 4-5 layers with tangentially-radially elongated cells (Table 4 and Fig. 6).

Trichomes characteristics
Two main types of trichomes are observed on the stem, petiole, and leaf blade; non-glandular and glandular.The non-glandular trichomes can be divided into unicellular, bicellular, multicellular, and dendritic trichomes with various stalk cell numbers.The glandular trichomes with unicellular stalk and multicellular head, Fig. 7.This result agrees with 50 .The density of trichomes is more abundant in W. obtusifolia.
While 6 asserts that Withania species differ in their morphological characteristics and 49,81 viewed the relevance of anatomical traits in distinguishing Withania species; stem with a circular outline in W. obtusifolia while circular with shallow ridges and furrows in W. somnifera and the number of vascular bundles; 14-15 in W. obtusifolia while 17-19 in W. somnifera, ovate petiole without ears in W. obtusifolia even though cat-face with ears in the other species, and leaf with tangentially elongated spongy cells in W. obtusifolia while radially elongated in W. somnifera.Other features have a limited role in characterizing the studied species which agrees with 46 who claims that it is difficult to differentiate them by anatomical features.

Protein and DNA profiles
The SDS-protein patterns of Withania obtusifolia and Withania somnifera allowed the identification of 5-7 major bands per lane, within molecular weights (Mwt) ranging from 20 to 67 Da (Table 5 and Fig. 8).Table 5 represented the Mwt, and the rate of flow (RF) of the formed bands of the two Withania plants.The highest Mwt (> 60 Da) and the lowest Mwt proteins (20 Da) were recorded in both plants.Five protein bands appeared to be conserved in both plants.The highest amounts of protein bands were recorded in W. obtusifolia showing the appearance of two new protein bands (at about 34 Mwt and 47 Mwt).Similarities and dissimilarities of protein bands between the two Withania plants were inscribed in Table 6.Results revealed that W. obtusifolia differed from W. somnifera by inducing 28.57% polymorphism.The SDS-Dendrogram (Fig. 9) clarified the subordination relationship of the proteins obtained from the two W. somnifera plants.SDS-Dendrogram declared that the Jaccard's similarity coefficient of the cluster analysis of the 2 plants was 0.5, which indicated that W. obtusifolia was moderately related to W. somnifera.Scot-DNA analysis was used to evaluate and compare the genetic variation of the two plants and was summarized in Tables 7 and 8 and Fig. 10.Table 7 revealed the generation of 96 total bands via the usage of 11 primers with an average of 70.83% polymorphism per primer.Scot-24, Scot-31, and Scot-52 primers possessed complete discrimination ability between the two Withania sp.Also, Scot-70 and Scot-34 primers revealed the highest differentiation potentiality with 9 (81.82%) and 8 (80%) polymorphic bands, respectively.However, Scot-71 has the lowest discrimination ability as it exhibited 4 polymorphic bands (40%) between the plants.In addition, the other employing primers produced special banding patterns ranging from 3-7 amplicons [3 (using Scot-61), 4 (using Scot-66), 5 (using Scot-33), 6(using Scot-13), and 7 (using Scot-14)].Similarities and dissimilarities of Scot-DNA-bands between the two plants were elucidated in Table 8 and Fig www.nature.com/scientificreports/obtusifolia generated more polymorphic bands than those of W. somnifera, recording 36 (72%) and 32 (69.57) polymorphic bands, respectively.Also, both Withania plants owned the same conserved monomorphic bands (14).The Scot-DNA-Dendrogram illustrated the hierarchical relationship of the DNA-banding obtained from the two plants via 11 primers, Fig. 11.The Dendrogram manifested that the Jaccard's similarity coefficient of the cluster analysis of the two plants was 0.357, which indicated that W. obtusifolia was poorly related to W. somnifera.
In the present study, SDS-protein results revealed that W. obtusifolia had both conserved and additional proteins in its profile, whereas W. somnifera had only conserved proteins (Tables 5 and 6).Scot-DNA analyses confirm the same protein trend for DNA that W. obtusifolia produced more polymorphic bands while retaining monomorphic ones (Tables 7 and 8).www.nature.com/scientificreports/According to 41 , there are intraspecific variations and polymorphism phenomena in Solanaceae.Because of their coexistence in a mixed population, the two morphologically similar species W. obtusifolia and W. somnifera were frequently misinterpreted.

Characters
Genetic studies are essential for studying inter and intra-species genetic variability, in which the use of protein profiles and molecular markers are powerful and useful tools.Different studies worked on assessing the genetic variation of W. somnifera plants.Indian W. somnifera plants were genetically assessed using SDS-PAGE and RAPD markers 82 , RAPD and AFLP markers 83 , RAPD and ISSR 57,84 , RAPD 55,85,86 , ISSR 87,88 and EST-SSR 89 , however; the Egyptian genotype was assessed with only RAPD marker by 90 .All those studies suggest the valuableness of using SDS-PAGE and molecular markers in detecting variation among W. somnifera plants.No studies were recorded on assessing the genetic variation of W. obtusifolia using protein and molecular markers analyses until 77 , who analyzed the interspecific relationship between Withania obtusifolia and Withania somnifera using www.nature.com/scientificreports/morphological, anatomical, and phytochemical identification in association with SDS-PAGE and RAPD analyses and found considerable variations between both species.Finally, this investigation found that morphological (Table 3 and Figs. 1, 3, 4), and anatomical (Table 4 and Figs. 5, 6) identifications of the two species highlight slight variation between these two species, while protein (Tables 5, 6 and Fig. 8), molecular analyses (Tables 7, 8 and Fig. 10) and phylogenetic analyses (Figs. 9, 11) showed considerable genetic variations between these two species.Those findings confirmed the whole divergences found between W. obtusifolia and W. somnifera.

Conclusion
In highlight of geographical, morphological, anatomical, pollen, and seed characteristics, the SDS-PAGE and Scot-PCR analyses of W. obtusifolia and W. somnifera confirmed the significant genetic divergence between these two species highlighted by their taxonomic analyses.Therefore, this study provided evidence that SDS-PAGE and Scot-PCR-based molecular analyses can be used as efficient tools for detecting and confirming similarity and phylogenetic relationships among the genus Withania.

Fig. 1 .
Fig. 1.Field photographs showing the morphological characters of Withania obtusifolia and W. somnifera respectively: (a, b) Twigs with leaves, flowers and fruits; (c, d) Flower buds; (e, f) Flower; (g) Sepal deltoid-ovate with a cushion in W. obtusifolia; (h) Sepal deltoid-lanceolate without a cushion in W. somnifera; (i) filament base without pillow-like in W. obtusifolia; (j) filament base with pillow-like in W. somnifera; (k, l) anther; (m) gynoecium with clavate stigma in W. obtusifolia; (n) gynoecium with bi-lobed stigma in W. somnifera; (o, p) plant with immature fruit; (q) orange red mature fruit in W. obtusifolia; (r) bright red mature fruit in W. somnifera; (s, t) 4-5 pitted seed in W. obtusifolia; (t) one pitted in W. somnifera.Photographs of W. obtusifolia were taken by Bernadette Simpson and those of W. somnifera were taken by Prof. Dr. Rim Hamdy.

Fig. 2 .
Fig. 2. Distribution map of Withania species in Egypt showing specimens of W. obtusifolia and W. somnifera examined by the authors.

Table 1 .
Collections data of the studied Withania species in Egypt.
Perennial herbs or shrubs with woody texture.Stems are erect, heavily branched with dense hairs.Leaves are solitary or paired, simple, alternate and petiolated.The leaf blade is entire, symmetrical-asymmetrical, Leaf ovate-elliptic with obtuse apex and truncate base.Inflorescence 1-6(8) flowers, calyx with tube longer than lobe, calyx lobe ovate.The Corolla tube is longer than the lobe.Filament without cushions at the base.Stigma clavate.Fruit orange-red with 23-25 seeds and seed diameter 1.8-2.2× 1.6-2 mm.

Table 2 .
Eleven Scot Primer sequences used in the study.

Table 3 .
Morphological characters within the studied Withania species (significant differences are in italics).

Table 4 .
The anatomical characteristics of the stem, leaf and petiole of the examined Withania species.

Table 5 .
The SDS-PAGE pattern of W. obtusifolia and W. somnifera showing molecular weight (Mwt) and the rate of flow (RF) of the formed bands.

Table 6 .
Protein band similarities and dissimilarities prompted by W. obtusifolia and W. somnifera.

Table 7 .
Scot analyses of W. obtusifolia and W. somnifera using eleven primers.

Table 8 .
Scot band similarities and dissimilarities prompted by W. obtusifolia and W. somnifera.